The broad goal of this project is to elucidate, in biochemical and cellular terms, a mechanism for regulated protein sorting within the endocytic pathway. The focus of the work in the upcoming funding period is on the export of cargo proteins from endosomes, a poorly characterized set of retrograde transport pathways that sort cargo to the Golgi apparatus. For some nutrient transporters, such as the yeast Fet3/Ftr1 reductive iron transporter, this step constitutes the initial stage of an endocytic recycling pathway that returns them to the plasma membrane for re-use. Model cargo proteins, including nutrient transporters, an acidic hydrolase sorting receptor, and SNARE proteins have been established to investigate these pathways and the genetic requirements for their trafficking have been determined. Proposed experiments will identify the structural features of cargo proteins that are necessary for retrograde sorting, and identify the components of the sorting machinery that recognize these signals. The research will focus on the roles of the retromer cargo sorting complex, an evolutionarily conserved complex of five proteins that are associated with endosome membranes, and sorting nexins, which are proposed to function as cargo receptors in endosome export pathways. Methodologies to be used include genetic analysis of trafficking in yeast mutant strains, kinetic analysis of protein turnover rates, biochemical characterization of interactions between cargo and the sorting machinery and between components of the sorting machinery. The studies will provide key insights into how cargo proteins are recognized in the endosomal system, how recognition is translated into export of cargo from endosomes, and how these molecular interactions are harnessed by the cell to modulate the composition of the plasma membrane. PUBLIC HEALTH RELEVANCE: Many aspects of cell physiology rely directly on the protein and lipid composition of the plasma membrane: nutrient acquisition, cell-cell recognition and signaling, cell-substrate adhesion, and cell motility. The molecular components of the plasma membrane are in a dynamic equilibrium with intracellular endosomes. The proposed studies will investigate mechanisms of protein sorting within endosomes that lead to their recycling back to the plasma membrane.